IMMUNOLOGY AND HOST-PARASITE INTERACTIONS - ORIGINAL PAPER

Besnoitia besnoiti–driven endothelial host cell cycle alteration

Zahady D. Velásquez1 & Sara Lopez-Osorio1,2& Learta Pervizaj-Oruqaj3,4,5 &

Susanne Herold3,4,5& Carlos Hermosilla1 & Anja Taubert1

Received: 15 January 2020 /Accepted: 1 June 2020# The Author(s) 2020

AbstractBesnoitia besnoiti is an important obligate intracellular parasite of cattle which primarily infects host endothelial cells of bloodvessels during the acute phase of infection. Similar to the closely related parasite Toxoplasma gondii, B. besnoiti has fastproliferating properties leading to rapid host cell lysis within 24–30 h p.i. in vitro. Some apicomplexan parasites were demon-strated to modulate the host cellular cell cycle to successfully perform their intracellular development. As such, we recentlydemonstrated that T. gondii tachyzoites induce G2/M arrest accompanied by chromosomemissegregation, cell spindle alteration,formation of supernumerary centrosomes, and cytokinesis impairment when infecting primary bovine umbilical vein endothelialcells (BUVEC). Here, we follow a comparative approach by using the same host endothelial cell system for B. besnoiti infections.The current data showed that—in terms of host cell cycle modulation—infections of BUVEC by B. besnoiti tachyzoites indeeddiffer significantly from those by T. gondii. As such, cyclin expression patterns demonstrated a significant upregulation of cyclinE1 in B. besnoiti–infected BUVEC, thereby indicating parasite-driven host cell stasis at G1-to-S phase transition. In line, themitotic phase of host cell cycle was not influenced since alterations of chromosome segregation, mitotic spindle formation, andcytokinesis were not observed. In contrast to respective T. gondii–related data, we furthermore found a significant upregulation ofhistone H3 (S10) phosphorylation in B. besnoiti–infected BUVEC, thereby indicating enhanced chromosome condensation tooccur in these cells. In line to altered G1/S-transition, we here additionally showed that subcellular abundance of proliferating cellnuclear antigen (PCNA), a marker for G1 and S phase sub-stages, was affected by B. besnoiti since infected cells showedincreased nuclear PCNA levels when compared with that of control cells.

Keywords Besnoitia besnoiti . Apicomplexan parasites . Coccidia . Cell cycle arrest . Histone H3 S10

Introduction

The apicomplexan obligate intracellular protozoa Besnoitiabesnoiti represents a coccidian parasite of major importance

in cattle industry. B. besnoiti infection was classified as an“emerging disease” in Europe by the European Food SafetyAuthority in 2010 (EFSA). Bovine besnoitiosis leads to severeskin alterations, vulvitis, vaginitis, orchitis, and infertility ofbulls and cows among other signs (Gollnick et al. 2018).Consequently, this parasite causes significant losses in com-mercial cattle industry and impairs individual animal welfare(Dubey and Lindsay 1996; Dubey 2003; Cortes et al. 2014).

It is well-known that apicomplexan parasites significantlymodulate their host cells to guarantee successful intracellulardevelopment and proliferation. As such, they influence nu-merous host cellular pathways, such as apoptosis, autophagy,cytoskeleton, metabolism, or immune reactions. In this con-text, some reports have indicated that tachyzoites of T. gondiidysregulate the host cellular cell cycle (Brunet et al. 2008;Molestina et al. 2008; Velásquez et al. 2019). The cell cycleof mammalian cells represents a highly regulated and complexprocesses that includes successive progression of distinct cell

Section Editor: Panagiotis Karanis

* Zahady D. Velásquezzahady.velasquez@vetmed.uni-giessen.de

1 Institute of Parasitology, Biomedical Research Center Seltersberg,Justus Liebig University, Schubertstr. 81, 35392 Giessen, Germany

2 Research Group CIVAB, School of Veterinary Medicine, Faculty ofAgrarian Sciences, University of Antioquia, Medellín, Colombia

3 Cardio Pulmonary Institute (CPI), Giessen, Germany4 Universities Giessen & Marburg Lung Center (UGMLC),

Giessen, Germany5 German Center for Lung Research (DZL), Giessen, Germany

https://doi.org/10.1007/s00436-020-06744-x

/ Published online: 17 June 2020

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cycle phases (G0-G1; S, and G2-M), which finally leads tocell division via cytokinesis. The cell cycle begins with theG1-phase (Gap-phase 1). In this step, the cell synthetizesmRNA and proteins that the next cell cycle steps. Afterward,the cell triggers the DNA synthesis machinery to duplicate itscomplete genome, in the so-called S-phase. Once this processis completed, the cell enters into a new process of growing andsynthetizing proteins, called the G2-phase. Finally, the cellactivates the genome division process, called mitosis, whichwill give rise to two daughter cells with the same genomecomposition and size (M-phase and cytokinesis). The transi-tion to each phase is tightly regulated by specific checkpointproteins and is based on sequential activation or inactivationof cyclins, cyclin-dependent kinases (Cdk), and cyclin-dependent kinase inhibitors (CDK-inhibitor). For instance,G1-phase is regulated by D- and E-type cyclins, while S-phase is controlled by A-type cyclins and G2/M-phase A-type and B-type cyclins (Vermeulen et al. 2003). Cyclin andits CDK partner modulates an intracellular signal that allowsfor the cell cycle progression. On the contrary, CDK-inhibitors regulate the cyclins-CDK complex activity and/ordegradation to allow the correct cell cycle development.

In case of protozoan infections, data indicate a species-specific host cellular cell cycle dysregulation. As such,T. gondii and Leishmania spp. induce cell cycle arrest andeventually dampen host cell proliferation (Brunet et al.2008; Costales et al. 2009; Kim et al. 2016; Kuzmenok et al.2005; Molestina et al. 2008; Scanlon et al. 2000; Velásquezet al. 2019), while Theileria annulata and Theileria parvatrigger host cell division and proliferation (von Schubertet al. 2010; Wiens et al. 2014) and induce segregation ofTheileria merozoites to each developing daughter cell.Conversely, L. amazonensis interferes early in cell cycle byG0/G1-phase arrest (Kuzmenok et al. 2005). In contrast,Plasmodium falciparum infections of HepG2 cells affect mi-tosis and lead to a binucleated phenotype and a lack of celldivision (Hanson et al. 2015). In the case of T. gondii, avail-able data record different modes of action, on one side, aninfection-driven shift from G0/G1 to S phase with an accumu-lation of host cells in S phase (Molestina et al. 2008; Lavineand Arrizabalaga 2009). On the other side, a host cellulararrest in the G2 phase (Brunet et al. 2008) or even both(Kim et al. 2016), thereby most probably reflecting celltype–specific reactions. We recently reported that T. gondiiinfections of primary bovine umbilical vein endothelial cells(BUVEC) lead to a G2/M arrest and trigger severe defectsduring mitosis as propagated by chromosome missegregation,supernumerary centrosome formation, and cytokinesis impair-ment (Velásquez et al. 2019). Given that no data exist onB. besnoiti–triggered host cell cycle modulation, and also togenerate comparative data with T. gondii, we here used thesame type (BUVEC) for B. besnoiti infections in order toreplicate in vivo infections as closely as possible and analyzed

the impact of this obligate intracellular parasite on cell cycleprogression. We here show for the first time that B. besnoitiinfection indeed alters cell cycle–related molecules (e.g., cy-clin E1, p27-kip1) but differs in its effects from T. gondii,therefore modulating host endothelial cell cycle progressionin a parasite species-specific manner.

Material and methods

Primary bovine umbilical vein endothelial cellisolation and maintenance

Primary bovine umbilical vein endothelial cells (BUVEC)were isolated from umbilical veins obtained from calves bornby sectio caesarea at the Justus Liebig University Giessen.Therefore, umbilical cords were kept at 4 °C in 0.9%HBSS–HEPES buffer (pH 7.4; Gibco, Grand Island, NY,USA) supplemented with 1% penicillin (500 U/ml; Sigma,St. Louis, MO, USA) and streptomycin (500 μg/ml; Sigma)for a maximum of 16 h before use. For the isolation of endo-thelial cells, 0.025% collagenase type II (WorthingtonBiochemical Corporation) suspended in Pucks solution(Gibco) was infused into the lumen of ligated umbilical veinsand incubated for 20 min at 37 °C in 5% CO2 atmosphere.After gently massaging the umbilical veins, the cell suspen-sion was collected in cell culture medium and supplementedwith 1 ml fetal calf serum (FCS, Gibco) in order to inactivatecollagenase. After two washes (350×g, 12 min, RT), cellswere resuspended in complete endothelial cell growth medi-um (ECGM, PromoCell, supplemented with 10% FCS), plat-ed in 25-cm2 tissue plastic culture flasks (Greiner) and kept at37 °C in 5% CO2 atmosphere. BUVEC were cultured in mod-ified ECGMmedium (EGCM, diluted at 30% in M199 medi-um, supplemented with 5% FCS (Greiner) and 1% penicillinand streptomycin) with medium changes every 2–3 days.BUVEC cell layers were used for infection after 3 passagesin vitro. All bovine primary endothelial cells samples wereconducted in accordance with the Institutional EthicsCommission of Justus Liebig Universität of Gießen(Germany), and in accordance with the current EuropeanAnimal Welfare Legislation: ART13TFEU.

Parasite tachyzoite maintenance

Tachyzoites of Besnoitia besnoiti (strain Bb1Evora03) weremaintained by serial passages either in primary bovine umbil-ical vein endothelial cells (BUVEC) or African green monkeykidney epithelial cells (MARC-145) according to (MuñozCaro et al. 2014). Confluent BUVEC monolayers in 25-cm2

flasks were infected with 2.5 × 105 freshly isolated B. besnoititachyzoites. From 48 h p.i. onwards, free-released viabletachyzoites were collected fromBUVEC culture supernatants,

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filtered through a 5-μm syringe filter (Sartorius AG), washedin modECGM and pelleted (400×g, 12 min). Tachyzoiteswere counted in a Neubauer chamber and suspended inmodECGM until further experimental use.

Protein extraction

Proteins from infected and non-infected BUVEC were ex-tracted by cell sonication (20 s, 5 times) in RIPA buffer(50 mM Tris-HCl, pH 7.4; 1% NP-40; 0.5% Na-deoxycholate; 0.1% SDS; 150 mM NaCl; 2 mM EDTA;50 mMNaF, all Roth) supplemented with a protease inhibitorcocktail (Sigma-Aldrich). Cell homogenates were centrifuged(10,000×g, 10 min, 4 °C) to sediment intact cells and nuclei.The protein content of RIPA buffer-soluble cell supernatantwas quantified via Coomassie Plus (Bradford) Assay Kit(Thermo Scientific) following the manufacturer’s instructions.Same protocol was applied for the tachyzoites protein extractproduction.

Cell nuclei extraction

Four BUVEC isolates were seed in T-25 flasks and infectedwith B. besnoiti (MOI 1:5). For controls, non-infected cellswere used. After 24 h p.i., cells were detached by a cell scraper(Thermo Fisher Scientific) and collected in 500 μL fraction-ation buffer (20 mM HEPES, pH 7.4, 10 mM KCL, 2 mMMgCl2, 1 mMEDTA, 1 mMEGTA, 1mMDTT, and proteaseinhibitor cocktail, Thermo Fisher Scientific). Cells were incu-bated for 15 min on ice, passed 10 times through a 27-gaugeneedle and kept on ice for 20 min. To separate nuclear andcytoplasmic fractions, samples were centrifuged at 720×g for5 min. The pellet (nuclear fraction) was washed thrice in500 μl fractionation buffer, and thereafter, nuclei weredisrupted by passing through a 25-gauge needle (10x).Nuclear proteins were then obtained by centrifugation at700×g for 10 min.

SDS-PAGE and immunoblotting

For immunoblotting, samples were supplemented with 6 Murea. After boiling (95 °C, 5 min), total proteins (60 μg/slot)were separated in polyacrylamide gels (12% or 15%) via elec-trophoresis (100 V, 1.5 h; tetra system, BioRad). Proteinswere transferred to polyvinylidene difluoride (PVDF) mem-branes (Millipore) (300 mA, 2 h). Blots were blocked in 3%BSA in TBS (50 mM Tris-Cl, pH 7.6; 150 mMNaCl contain-ing 0.1% Tween (blocking solution); Sigma-Aldrich) for 1 hat RT and then incubated in primary antibodies (see Table 1)diluted in a blocking solution (overnight, 4 °C). Detection ofvinculin was used as loading control for normalization ofsamples. Following three washings in TBS-Tween (0.1%)buffer, blots were incubated in adequate secondary antibody

solutions (diluted in blocking solution, for dilution: seeTable 1; 30 min, RT). Following three further washings inTBS-Tween buffer, signal detection was accomplished by anenhanced chemiluminescence detection system (ECL® pluskit, GE Healthcare) and recorded using a ChemoCam Imager(Intas Science Imaging). Protein masses were controlled by aprotein ladder (PageRuler Plus® Prestained Protein Ladder ~10–250 kDa, Thermo Fisher Scientific). Protein band intensityquantification was analyzed using a Fiji Gel Analyzer®plugin. For all protein analyzed, the parasitic homologousprotein was detected (data not shown).

Immunofluorescence assays

Cell layers were fixed with paraformaldehyde (4%, 15 min,RT), washed thrice with PBS and incubated in blocking/permeabilization solution (PBS with 3% BSA, 0.1% saponin;1 h, RT). Thereafter, samples were incubated in primary anti-bodies (see Table 1) diluted in blocking/permeabilization so-lution (overnight, 4 °C, in a humidified chamber). After threewashings in PBS, samples were submitted to secondary anti-body solution (see Table 2; 30 min, RT, darkness). Cell nucleiwere labeled with 4′,6-diamidin-2-phenylindol (DAPI) beingpresent in mounting medium (Fluoromount G, ThermoFisher,495952).

Flow cytometry–based analysis of cell cycle phases

Cellular DNA content was measured using the FxCycle Far®red stain reagent (Invitrogen, F10348) according to the man-ufacturer’s instructions. The samples were analyzed with aFACSCalibur® Analyzer (Becton-Dickinson, Heidelberg,Germany) applying 633/5 nm excitation and emission collect-ed in a 660/20 bandpass. Cells were gated according to theirsize and granularity. Exclusively morphologically intact cellswere included in the analysis. Data analysis was performed by

Table 1 Primary antibodies used for western blot and immunofluorescence

Primary antibodies

Antigen Company Cat. number Origin Dilution

Vinculin Santa Cruz sc-73614 Mouse 1:1000

Cyclin A2 Abcam ab39 Mouse 1:1000

Cyclin B1 Abcam ab32053 Rabbit 1:3000

Cyclin B1 S126 Abcam ab133439 Goat 1:1000

Cyclin E1 Abcam ab133266 Rabbit 1:1000

Histone H3 S10 Abcam ab5176 Rabbit 1:1000

PCNA Abcam ab18197 Rabbit 1:1000

α-Tubulin Thermo Fisher A11126 Mouse 1:100

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the use of the FlowJo® (version 10.5.0) flow cytometry anal-ysis software (FlowJo LLC, Ashland, OR).

Confocal microscopy

All immunofluorescence analyses were performed by confo-cal microscopy (× 63 magnification with a numerical apertureof 1.4, LSM 710, Olympus). Two types of image acquisitionwere used: (i) multi-channel images which were merged todefine the co-localization of the signal, and (ii) Z-stacks of0.3–0.5 μm for cell spindle and chromosome detection.Image processing was carried out by Fiji ImageJ® using Z-projection and merged-channel-plugins being restricted tooverall adjustment of brightness and contrast.

Live cell 3D-holotomographic microscopy

3D-holotomographic images were obtained by using 3D CellExplorer microscope (Nanolive® 3D) equipped with an × 60magnification optic (λ = 520 nm, sample exposure 0.2 mW/mm2) and a field depth of 30μm. Images were analyzed usingthe STEVE® software (Nanolive) to obtain a refractive index-based z-stack. Digital staining was applied according to therefractive index of intracellular structures. Nuclei were stainedby DRAQ5 probe for vital DNA staining following the man-ufacturer instructions (DRAQ5™ Fluorescent Probe Solution,ThermoFischer).

Statistical analysis

The data from the total cell number in the monolayer and forWB assay were expressed as mean ± SEM from six BUVECisolates. For cell number– and FACS-based assays, one-wayanalysis of variance (non-parametric ANOVA) with Kruskal-Wallis post-test was performed using the GraphPad Prism® 7software applying a significance level of 5%. Forimmunoblot-based analyses, unpaired two-tailed T tests wereperformed with Mann-Whitney post-test comparing controlsvs infected cells, with a 95% confidence interval.

Results

B. besnoiti infections do not affect host cellproliferation

Even though B. besnoiti tachyzoites were recorded to success-fully replicate in various immortalized host cell types (Corteset al. 2007; Samish et al. 1988; Shkap et al. 1991), the kineticof parasite divisionmay differ depending on the host cell typesinfected in vivo. For this reason, we first estimated B. besnoitidivision cycles in primary BUVEC by analyzing the numberof tachyzoites present in each parasitophorous vacuole (PV) ata different time of infection (Fig. 1a). Using an MOI of 5:1 inthree BUVEC isolates, we achieved an infection rate of 46.23± 14.73%. Overall, we observed a non-synchronous prolifer-ation of tachyzoites in BUVEC. First, tachyzoite division(detection of 2-mers) was observed at 6 h p.i.; from thereonwards, an ongoing division was estimated and mosttachyzoites had divided at 18 h p.i. (Fig. 1a). At 24 h p.i., 1-to 32-mers were found in intracellular PVs (Fig. 1b).

The effect ofB. besnoiti infections on host cell proliferationwas examined by counting total BUVEC numbers. Given thatwe worked with a primary cell type, considerable variations inthe size of cells and the time of cell division can be expectedbetween isolates. Taking this into account, we included sixbiological replicates and used identical cell numbers forseeding. Given that B. besnoiti belongs to fast-replicatingcoccidian parasites and host cell lysis mainly occurs from24 h p.i. onwards (thereby potentially inducing artifacts inhost cell enumeration), we here restricted all host cellproliferation–based analyses to 1 day p.i. The determinationof total host cell numbers revealed no statistically significantinfluence of B. besnoiti infections on host cell proliferationwhen compared with non-infected BUVEC controls (Fig.1b–c). Nevertheless, the analysis of each individual BUVECisolate after 24 h p.i., which showed a decreasing tendency inthe number of cells in the monolayer (Fig. 1c). This evidencewas considered important because all samples, were seededthe same number and at the same time. Overall, four isolatesfrom a total of six showed a decrease of the total amount ofcells in the monolayer after 24 h p.i. Inter-donor variation in

Table 2 Secondary antibodiesused for western blot andimmunofluorescence

Secondary antibodies

Name Company Cat. number Reactivity Dilution

Goat anti-mouse IgG Peroxidase conjugated Pierce 31430 Mouse 1:40000

Goat anti-rabbit IgG peroxidase conjugated Pierce 31460 Rabbit 1:40000

AlexaFluor 488 Thermo Fisher A11008 Rabbit 1:500

AlexaFluor 488 Thermo Fisher A11001 Mouse 1:500

AlexaFluor 594 Thermo Fisher R37117 Rabbit 1:500

AlexaFluor 594 Thermo Fisher A11005 Mouse 1:500

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different parameters between BUVEC isolates in different ex-perimental set-ups had been observed before, and, thus, wemade efforts to interpret the variation on a case by case basis(Conejeros et al. 2019; Taubert et al. 2016).

We recently showed that T. gondii infections resulted in asignificantly enhanced proportion of BUVEC presentingmorethan one nucleus (mainly binucleated BUVEC; Velásquezet al. 2019). For comparative reasons, we here also estimated

the cell nucleus numbers in B. besnoiti–infected BUVEC andnon-infected controls. Our results showed that B. besnoiti in-fections had no effect on host cell nucleus numbers (Fig. 1d).This was also tested via DRAQ5 (vital staining of nuclei)-based live cell 3D holotomographic microscopy (3DNanolive®). By this novel live cell imaging technique, weshowed that B. besnoiti infections did not affect the nuclearphenotype, while T. gondii infections, which were used for

Fig. 1 Infection of BUVEC with B. besnoiti tachyzoites and effect ofinfection on host cell proliferation. Sub-confluent BUVEC were infectedwith B. besnoiti tachyzoites at an MOI 5:1 and analyzed after 24 hpi. aQuantification of tachyzoites numbers/parasitophorous vacuole during30 h of infection. The mature structures were observed between 24 and30 h p.i. After this time point, all parasites were released. b Illustration ofnon-infected control cells and B. besnoiti–infected BUVEC at 24 h p.i. cHost cell proliferation analyzed by estimating cell numbers of

B. besnoiti–infected BUVEC and non-infected controls at 24 h p.i. Nostatistically significant differences were observed, but it was a hint ofdecreased number of cells at the B. besnoitia–infected monolayer. dQuantification of binucleated host cells in B. besnoiti–infected BUVECand non-infected controls. e Illustration of DRAQ5 (vital DNA staining,red)-stained host cell nuclei in B. besnoiti– and T. gondii–infectedBUVEC (24 h p.i.) via 3D holotomographic microscopy (note: binucle-ated phenotype in case of T. gondii–infected BUVEC)

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comparative reasons, indeed induced a binucleated phenotypein BUVEC (Fig. 1e).

B. besnoiti induces host cell stasis at G1-to-S-phasetransition

To estimate whether B. besnoiti infections dysregulate hostcellular cell cycle progression in BUVEC, we performed flowcytometry–based analyses on the cellular DNA content. Thiswell-established method simply measures cellular DNA abun-dance, thereby allowing for the discrimination of the threemain periods of the cell cycle (G0/G1-; S-; G2/M-phase).Even though this technique lacks high sensitivity, we couldrecently demonstrate a T. gondii–driven shift of cell cyclephases in BUVEC by applying this method (Velásquez et al.2019). Here, the analysis was performed on BUVEC

population in the SSC-H vs FSC-H graph (exemplary illustra-tion in Fig. 2a), where we selected the three main peaksrepresenting the three main phases of the cell cycle (G0/G1-;S-; G2/M-phase). However, by comparing total cell layers ofB. besnoiti–infected BUVEC with non-infected ones, wecould not identify any influence of B. besnoiti infections onhost cell cycle progress (Fig. 2b). Nevertheless, the G1-phaseof infected cells seemed to be higher in comparison with thenon-infected monolayer. On account of this, we analyzed G0/G1-phase in more detail (Fig. 2c).We conducted a correlationbetween the value of each BUVEC isolate in the non-infectedcondition in comparison with the value of the same isolate, butin the B. besnoitia–infected layer. The results showed that 4/6isolates revealed an increased number of cells in the G0/G1-phase (isolates 1–3, 5), 1/6 was unchanged (isolate 4), and 1/6showed a decrease in the percentage of cells in G0/G1 (isolate

Fig. 2 Distribution of cell cycle phases in B. besnoiti–infected BUVEC.BUVECwere infected with B. besnoiti and examined for DNA content at24 h p.i. by FACS-based analyses. a Flowchart of the FACS analysisshowing the total number of cells in G- (one genomic DNA copy), G2-(two genomic copies), and S- (the cell population in between both phases)phase. The cells were first gated to eliminate debris from the analysis.Furthermore, the DNA channel vs the population histograme was used to

get the total number of cells in each peak. b Mean data obtained fromB. besnoiti–infected BUVEC (n = 6), plotted as a percentage of the totalcells vs DNA amount. Bars represent the median ± SD. cG1-phase graphfrom (b) comparing the BUVEC isolates distribution between non-infect-ed and B. besnoiti-infected cells. Light increase in the number of cells wasobserved in the peak corresponding toG0/G1; however, the valuewas notstatistically significant

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6). Given that analyses based on rough cellular DNA contentmay not be sensitive enough to detect minor changes in cellcycle progression, we additionally controlled B. besnoiti–infect-ed BUVEC for protein abundance of several key molecules reg-ulating cell cycle phases. Thus, cyclin B1 (indicative for G2 toM-phase transition), cyclin E1 (indicative for G1/S phase

transition), and cyclin A2 (indicative for S/G2-phases transition)were controlled for expression via western blotting. Furthermore,p27-kip1 and p57-kip2 (inhibitors of G1-S transition cyclins)were analyzed in their abundance to control for a potential cellcycle arrest. Given that primary BUVEC monolayers vary con-siderably in their individual reactions, we here included five

Fig. 3 Analysis of cell cycle–related molecule expression inB. besnoitia–infected BUVEC. a Five biological replicates of BUVECwere infected with B. besnoiti tachyzoites and analyzed by western blot-ting for the abundance of the cell cycle–related molecules cyclin A2,cyclin B1, cyclin E, p27-Kip1, and p57-Kip2 at 24 h p.i. The density of

the protein signals was quantified, and graphed as a relative ratio tovinculin (housekeeping protein). Cyclin E1 and p27-kip1 were upregu-lated in infected cells thereby indicating a G0/G1 cell cycle arrest. Barsrepresent the median ± SD of five biological replicates

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different BUVEC isolates. Western blotting analyses revealedcyclin E1 to be significantly enhanced in its abundance inB. besnoiti–infected BUVEC when compared with non-infected control cells (p = 0.00314; Fig. 3). Due to a technicalproblem in the BUVEC isolate number 5, the measurementsfrom the non-infected and the B. besnoitia–infected cells wereerased from the statistical analysis. No significant changes weredetected for cyclin A2 or cyclin B1, suggesting that neither G2phase nor mitosis was affected by B. besnoiti infections. In con-trast to p57-Kip2, we furthermore detected a significant increaseof p27-Kip1 abundance in B. besnoiti–infected BUVEC(infected vs control cells, p < 0,0379; Fig. 3). This regulativemolecule is a cyclin-CDK inhibitor blocking cyclin E-CDK2

complex activation. Overall, current expression profiles sug-gested that B. besnoiti–infected cells experienced a stasis at G1-to-S phase transition.

B. besnoiti infections alter neither chromosomecondensation nor mitotic spindle formation in BUVEC

Since we recently showed that mitosis was impaired onthe level of chromosome condensation and mitotic spindleformation in T. gondii infections in BUVEC (Velásquezet al. 2019), and since the parasite may modulate host cellcycle on different functional levels, we additionally con-trolled whether B. besnoiti infections may also modulatemitosis progression. Correspondingly, both non-infectedand B. besnoiti–infected BUVEC were stained for chro-mosomes (DAPI), microtubules forming the mitotic spin-dle (γ-tubulin), and for centrosomes (γ-tubulin). In total,100 cells were analyzed for each condition. Confocal mi-croscopic analyses revealed that B. besnoiti infections nei-ther affected chromosome condensation nor mitotic spin-dle formation during mitosis (Fig. 4). As such, followingchromosome arrangement in infected mitotic host cells,we detected normal segregation of chromosomes in

Fig. 5 Analysis ofphosphorylated-histone H3 andPCNA expression inB. besnoitia–infected BUVEC. aFive biological replicates ofBUVEC were infected withB. besnoiti tachyzoites and ana-lyzed by western blotting forphosphorylated histone H3 (S10)and PCNA expression at 24 h p.i.The density of the protein signalswas quantified and graphed as arelative ratio to vinculin ashousekeeping protein. Both pro-teins were upregulated, but onlyp-HH3S10 was statistically sig-nificant. Bars represent the medi-an ± SD from five biological rep-licates. ** = p < 0.0741

�Fig. 4 Exemplary illustration of chromosome segregation and mitoticspindle formation in mitotic B. besnoiti–infected BUVEC. MitoticB. besnoiti–infected BUVEC and non-infected cells were stained withanti-pHH3 S10 (green) for chromosome detection, with anti-α-Tubulin(red) for mitotic spindle detection. Pictures were taken as a confocal series(z-stack) via confocal microscopy and lately processed as flat imagesusing the ImageJ software (z-projection). The mitotic spindle structureat each cell cycle phase demonstrated that B. besnoiti is not inteferingwith the mitosis process of the host cell. Scale bar represents 5 μm

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prophase, metaphase, anaphase, and telophase (Fig. 4-blue). Likewise, the formation and migration of thetubulin-based cytoskeleton corresponded well to the mi-totic spindle shape in control cells for each step (Fig. 4-red). Furthermore, no changes were detected in cases ofcentrosome formation and localization in mitotic stages(Fig. 4-green). However, when estimating the abundanceof phosphorylated histone H3 (Ser10; a classical chromo-some condensation marker), a significantly higher expres-sion level of this protein was found in B. besnoiti–infectedBUVEC when compared with that of control cells (infect-ed vs control cells, p < 0,0017; Fig. 5).

B. besnoiti infection induces an increase in nuclearPCNA abundance

Proliferating cell nuclear antigen (PCNA) is critically in-volved in DNA replication and repair, and has cell cycle–dependent properties. PCNA nuclear distribution is character-istic for G phase, early, mid-, and late S phase, and is thereforeoften used as a marker for sub-stages of DNA replication(Schönenberger et al. 2015). Given that B. besnoiti–infectedcells seemed to be arrested at G1/S transition, we here ana-lyzed S-phase progression by estimating PCNA localizationand nuclear/cytoplasmic abundance. When comparing totalPCNA expression in B. besnoiti–infected host cells and non-infected BUVEC layers via western blotting at 24 h p.i., nosignificant differences could be identified, indicating that nottotal PCNA abundance was not changed (Fig. 5). Given thattachyzoites exclusively reside in the cytoplasmic compart-ment, parasite-derived signals could falsify these data. Wethen separated nuclear and cytosolic fractions from bothnon-infected and B. besnoiti–infected host cells (24 h p.i.)for western blot analyses. The purity of each extract was con-firmed by the detection of typical cytosolic (α-tubulin) ornuclear (lamin B1) proteins. As expected, PCNA was mainlyexpressed in the nuclear fraction when compared with cyto-solic extracts. In line with our observations described above,the nuclear PCNA abundance inB. besnoiti–infected cells wasenhanced in nuclear extracts (which are devoid of parasites)(Fig. 6); however, this was not statistically significant (infect-ed vs non-infected cells, p = 0.1615). Neither total nor nuclearPCNA abundance showed significant differences when ana-lyzed byWB at 24 h p.i. However, by confocal microscopy, atime-dependent increase of nuclear PCNA abundance with amaximum at 12 h p.i. was observed (Fig. 7). We estimated thenuclear PCNA abundance during 24 h of in vitro infectionusing immunostaining (Fig. 7) and calculated PCNA signalintensity relative to the host cell nucleus (DAPI signal). Intotal, 300 cells were analyzed for each condition.B. besnoiti–infected cells showed a transient peak at 12 hp.i. (Fig. 7b, infected vs non-infected cells, at all-time pointstested, p < 0.0001), decreasing after 18 h p.i. The nuclear

PCNA localization showed the characteristic pattern for aG1-phase (Fig. 7a).

Discussion

B. besnoiti belongs to the apicomplexan cyst-forming proto-zoan Sarcocystidae family. During the acute phase of bovine

Fig. 6 PCNA abundance in nuclear and cytosolic protein extracts ofB. besnoiti–infected BUVEC. Nuclear and cytosolic protein extractsfrom four biological replicates of B. besnoiti–infected BUVEC andcontrols cells were subjected to western blotting to quantify PCNAexpression. For control of nuclear and cytosolic fraction purity, theabundance of α-tubulin (typical cytosolic protein) and lamin B1 (typicalnuclear protein) was analyzed. The density of the protein signals wasquantified and graphed as relative ratios: PCNA vs Lamin B1 for nuclearextracts and PCNA vs α-tubulin for cytosolic extracts. The ratio ofPCNA/vinculin showed higher values in comparison with the cytosolicone; however, it was not statistically significant. Bars represent the me-dian ± SD from five biological replicates

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besnoitiosis in vivo, tachyzoites continuously infect host endo-thelial cells of blood and lymphatic vessels, thereby replicatingfast intracellularly (Basson et al. 1970; Bigalke 1981; Muñoz-Caro et al. 2014). In contrast to the well-investigated and closelyrelated parasite T. gondii, no data are currently available on theinfluence of B. besnoiti infections on host cellular cell cycleprogression even though several reports indicated both species-and cell type–specific reactions in this respect (Maksimov et al.2016; Taubert et al. 2016). To demonstrate true species-specificreactions and to exclude host cell type–specific effects, we heretook advantage of recently published cell cycle–related data onT. gondii infections (Velasquez et al. 2019) by using the same

in vitro primary host cell system (bovine umbilical vein endothe-lial cells) under identical culture conditions, thereby standardiz-ing experimental conditions. Using this approach, we hereshowed that B. besnoiti indeed differentially modulates host en-dothelial cellular cell cycle when compared with T. gondii andaffects G1/S transition and relatedmolecules in a species-specificmanner.

B. besnoiti infections affect cattle as intermediate hosts and,as stated above, endothelial host cells of vessels represent themain host cells being infected during acute phase of infection(Basson et al. 1970; Bigalke 1981). In order to be rather closeto the in vivo situation and to allow a direct comparison with

Fig. 7 Exemplary illustration ofPCNA distribution inB. besnoiti–infected BUVEC. aB. besnoiti–infected BUVEC andcontrol cells were stained forDNA (DAPI, blue), B. besnoititachyzoites (in-house antiserum,red) and for PCNA (green) andanalyzed via confocal microsco-py. b Measurements of nuclearPCNA-related signals in relationto total cell-derived signals duringin vitro infection (4–24 h p.i.).The insets in 24 h p.i. picturesrepresent PCNA localization inthe non-infected cells nuclei at thesame time. The PCNA signalshowed a peak at 12 h p.i., beenprogressively decreased after 18 hp.i. Scale bar represents 5 μm.Asterisks: surface PCNA locali-zation in B. besnoiti tachyzoites.White arrows: PCNA signal in thenuclear compartment. Bars repre-sent the median ± SD against thenon-infected cells control. **** =p < 0.0001

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recently published T. gondii data (Velásquez et al. 2019), thecurrent study was performed in a primary bovine endothelialcell type. Furthermore, by using a primary host cell type, falseinfluences on host cellular cell cycle progression or cell divi-sion activities driven by cell immortalization or tumoral originwas avoided (Mondello and Chiodi 2013). It is worth notingthat we here exclusively analyzed host cell layers for up to24 h p.i. to avoid false-negative effects triggered by parasite-induced host cell lysis. This contrasts to experimental condi-tions of other studies on coccidian parasites, such as T. gondii,in which host cell proliferation was estimated for up to 48 hp.i. or even 120 h p.i. (Brunet et al. 2008).

By microscopical means, we confirmed that bovine endo-thelial cells (BUVEC) allowed full intracellular merogony de-velopment and resulted in the release of mature and motileB. besnoiti tachyzoites. Given that individual primary endo-thelial cell isolates generally show high inter-donor variability(Joyce 2003; Zhu and Joyce 2004), we here always includedat least five BUVEC isolates in each experimental setting.Consequently, we assume that observed differences in hostcell modulation in the current study are indeed realistic.

By estimating total BUVEC numbers at 24 h p.i., weshowed that B. besnoiti infections do not influence host cellproliferation. This finding contrasts with data on T. gondiiinfections which were reported either to dampen host cellproliferation in non-endothelial cell types (Brunet et al.2008; Molestina et al. 2008; Kim et al. 2016) or to triggerproliferation in the same endothelial cell type we used(Velásquez et al. 2019). Accordingly, we could also not statean infection-driven increase of bi- or multi-nucleated hostcells as previously reported for T. gondii infections(Velásquez et al. 2019). In this respect, the combination ofDRAQ5 staining with live cell 3D-holotomographic micros-copy easily allowed nuclear phenotype estimation and nicelyillustrated B. besnoiti development in BUVEC.

In contrast to T. gondii–related cell cycle data (Velásquezet al. 2019), FACS-based quantification of cellular DNA con-tents did not reveal B. besnoiti–driven changes in cell cyclephase patterns when compared with non-infected host cells.This may be based on different reasons: (i) this method is notsensitive enough to detect moderate changes in cell cyclephases, and (ii) when analyzing infected cell layers, this meth-od (which merely estimates crude DNA abundance) cannotdistinguish between host cell- and parasite-derived DNA.Given that we achieved a rather high infection rate with sev-eral meronts per host cell (2–20, which is typical for endothe-lial B. besnoiti infections in the current experimental set-up),the merge of parasite- and host cell–derived reactions mayhave masked alterations in host cellular cell cycle phases.Nonetheless, analysis of cell cycle–relatedmolecules involvedin regulation revealed cyclin E1 (one of the main regulatoryprotein of G1-phase and G1-to-S-phase progression) to beselectively upregulated in B. besnoiti–infected cell layers

since other cyclins (cyclin A2, cyclin B1, or the regulatorymolecule p57-kip2) did not show any altered abundance.Cyclin E1 represents an activator of cyclin-dependent kinase(CDK) 2, accumulates at G1/S boundary of cell cycle, andstimulates entry into and progression through S phase (Sauerand Lehner 1995; Ohtsubo et al. 1995; Ekholm and Reed2000). In somatic mammalian cells, cyclin E1 levels typicallydecline during S phase, thereby reaching low or undetectablelevels by the time replication is completed (Ekholm et al.2001). Thus, an enhancement or accumulation of cyclin Eindicates that B. besnoiti infection either triggers cyclin E1expression or blocks its degradation, which both affect thecellular exit from G1-phase. Another interesting finding wasthe upregulation of p27-kip1, a CDK inhibitor belonging tothe Cip/Kip family. The main regulation of p27-kip1 is medi-ated by proteolysis via ubiquitin/protease pathway at the G1/Sboundary (Lisztwan et al. 1998). This process is closely relat-ed with cyclin E1-CDK2 complex activity, which modulatesp27-kip1 phosphorylation and allows its entrance into theubiquitination pathway. Cells can only enter into S-phasewhen p27-kip1 proteolysis is completed. Thus, the currentdata suggested that B. besnoiti infection affected proper cyclinE1-CDK formation or activationwhich directly interferes withp27-kip1 proteolysis and is concomitant with G1-phase arrest.The finding of B. besnoiti–driven G1/S arrest was revealed asspecies-specific since T. gondii infections triggered stasis ofBUVEC in (G2/)M-phase (Velásquez et al. 2019).Furthermore, records on T. gondii–driven cell cycle impair-ment in non-endothelial cells reported an infection-drivenshift from G0/G1- to S-phase (Molestina et al. 2008; Lavineand Arrizabalaga 2009) or a host cellular arrest in G2-phase(Brunet et al. 2008), thereby most probably indicating celltype–specific reactions.

G1 phase is followed by S-phase, which signifies the stepduring which cells duplicate their genome for daughter cellformation. PCNA, which is involved in DNA replication, hascell cycle–dependent properties and typically localizes to nu-clear sites of active replication during S-phase (Bravo andCelis 1985; Celis and Celis 1985a; Essers et al. 2005;Sugimoto 2015). Thus, precise PCNA nuclear distribution isused to discriminate sub-stages of S phase (early, mid, and lateS phase) (Bravo and Celis, 1985; Celis and Celis 1985a, b;Ersoy et al. 2009). Given that the current data indicated aB. besnoiti induced at G1/S transition and assuming that mul-tiple phases may be targeted by the parasite, we here analyzedsubcellular PCNA abundance and distribution in B. besnoiti–infected BUVEC. One interesting finding, was that host cel-lular PCNA distribution changed throughout in vitromerogony, showing that even when PCNA was located inthe nuclear compartment, it showed a typical pattern of G1-phase after 18 h p.i. (homogeneous distribution in the nucle-us). Nevertheless, quantification of nuclear PCNA signals re-vealed an increase of PCNA abundance during B. besnoiti

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infection in BUVEC peaking at 12 h p.i., which was the lastpoint in time when some S-phase typical pattern could beobserved. Correspondingly, when preparing nuclear and cyto-plasmic extracts from B. besnoiti–infected BUVEC for west-ern blot–derived expression analyses, an increase of PCNAabundance was observed in infected BUVEC. Overall, thesedata suggest that B. besnoiti induces a G1 arrest in a time-dependent manner, thereby hampering host cells to enter intoS-phase.

Emerging data on chromosome formation suggest thatwhenever chromosomes fail to properly replicate in S-phase,subsequent segregation of sister chromatids during mitosiswill be impaired as well (Mankouri et al. 2013). For this rea-son and in order to compare B. besnoiti–driven reactions withT. gondii–related ones, we here also analyzed host cellularchromosome segregation, i.e., a cellular process that is highlyregulated and requires numerous factors for adequate process-ing, such as molecules mediating DNAmovement, DNA link-age to cellular structures, and chromosome maintenance(Duro and Marston 2015). Obviously, proper mitotic spindleformation, being based on tubulin-derived structures for chro-mosome cell linkage (Inoué and Salmon 1995; Inoué 1997;Rieder and Khodjakov 2003), is also fundamental for ade-quate chromosome segregation. Recently, we showed thatT. gondii infection of BUVEC highly interferes with theseprocesses and leads to chromosome missegregation and mi-totic spindle impairment (Velásquez et al. 2019). However,B. besnoiti–infected BUVEC showed normal chromosomestructures and mitotic spindle formation, and no impairmentof these processes could be stated, thereby indicating species-specific effects (once) again. Even though the mitotic processdid not seem to be affected in B. besnoiti infections, analyseson phosphorylated histone H3 (S10) showed a significantlyenhanced abundance in B. besnoiti–infected host cells. Todate, two functions have been assigned to serine 10 phosphor-ylation of histone H3 with one being related to chromosomecondensation and the other being linked to distinct gene tran-scription activation (Prigent and Dimitrov 2003). In bothcases, the serine phosphorylation–mediating kinase appearsfundamental (Schmitt et al. 2002; Taylor 1982; DeMannoet al. 1999; Cheung et al. 2000; Hans and Dimitrov, 2001).This process selectively occurs in some specific genome re-gions, depending on the stimuli that the cell received. It hasbeen previously identified that only some subsets of genes arealtered; one of these is related with the inflammatory responsetriggered by cytokines (Saccani et al. 2002). To date, distinctcytokine responses are well-known for T. gondii andP. falciparum infections (Baker et al. 2008; Fischer et al.1997), and inflammatory responses were also described forB. besnoiti infections in BUVEC, thereby allowing us to hy-pothesize that an increase of HH3 S10 phosphorylation drivenby B. besnoiti infection may also be explained by the inflam-matory response triggered by the parasite.

In conclusion, we here showed that B. besnoiti tachyzoiteinfection indeed affects host endothelial cell cycle progressionin a species-specific manner and avoids host cell cycle pro-gression by a G1-phase arrest. Further research will elucidatethe precise mechanisms, signaling pathways, and moleculeswhich are involved in this complex process.

Acknowledgments Authors thank A. Wehrend (Clinic for Obstetrics,Gynecology, and Andrology of Large and Small Animals, Justus LiebigUniversity Giessen, Giessen, Germany) for the continuous supply of bo-vine umbilical cords. Dr. Kalus Deckmann (Institut für Anatomy undZellbiologie, Justus Liebig Universität Giessen, Giessen, Germany) forproviding free access to a Zeiss LSM 710 confocal microscope. We aregrateful to Hannah Salecker for her valuable work in primary host cellisolation as well as parasite maintenance.

Author contributions ZDV and AT planned and designed the experi-ments. ZDV, SLO, and LPO performed the experiments. FACS readingand analysis: ZDV, LPO, and SH. ZDV, SLO, CH, and AT performedanalyses and interpretation of the data. All authors prepared, revised, andapproved the final version of the manuscript.

Funding Information Open Access funding provided by Projekt DEAL.

Compliance with ethical standards

Competing interests The authors declare that they have no competinginterests.

Open Access This article is licensed under a Creative CommonsAttribution 4.0 International License, which permits use, sharing, adap-tation, distribution and reproduction in any medium or format, as long asyou give appropriate credit to the original author(s) and the source, pro-vide a link to the Creative Commons licence, and indicate if changes weremade. The images or other third party material in this article are includedin the article's Creative Commons licence, unless indicated otherwise in acredit line to the material. If material is not included in the article'sCreative Commons licence and your intended use is not permitted bystatutory regulation or exceeds the permitted use, you will need to obtainpermission directly from the copyright holder. To view a copy of thislicence, visit http://creativecommons.org/licenses/by/4.0/.

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